Abstract
Hydrogen production from agro-industrial wastes is a sustainable alternative to fossil fuels. This study evaluated Escherichia coli wild type BW25113 and a septuple mutant ( ΔhyaB ΔhybC ΔhycA ΔfdoG ΔldhA ΔfrdC ΔaceE ) for biohydrogen production using mixtures of sugar beet pulp (SBP) and sugar beet molasses (SBM). Hydrolysates were pretreated, diluted (2 × , 5 × , 10 × ), and combined at different ratios (25/75, 50/50, 75/25), with or without glycerol. Undiluted substrates inhibited growth due to high phenolic and flavonoid content, while dilution restored fermentation. Molasses-rich mixtures (25/75) supported the highest hydrogen yields, reaching 759 NmL in the wild type (704.7 mL H 2 (g sugar −1 g biomass −1 ) and 1195 NmL in the mutant (979.5 mL H 2 (g sugar −1 g biomass −1 ). Biomass formation increased with dilution in the wild type, meanwhile the mutant strain was more sensitive to inhibitors and highest growth was observed in 5x diluted media. Results demonstrate that combining SBP and SBM provides synergistic advantages and that genetic engineering of E. coli enhances hydrogen metabolism, highlighting sugar beet by-products as promising substrates for efficient waste-based hydrogen production. • Molasses-rich mixtures produced 759 NmL H 2 in the wild type and 1195 NmL in the mutant. • Undiluted hydrolysates inhibited growth and hydrogen production due to high phenolic and flavonoid content. • Highest H 2 production was observed with the septuple mutant in SBP/SBM twice diluted with glycerol mixture.